We plan to investigate the endocrine mechanisms involved in the control of melanogenesis and growth of cultured melanoma cells. Because continued transcription and translation is necessary for MSH action, studies will be continued to determine if cAMP stimulates tyrosinase activity by causing de novo enzyme synthesis. Immunoprecipitation techniques will be utilized in these studies to quantitate the amount of tyrosinase synthesized in either control or MSH-treated cultures at various times after hormone administration. Because of our recent findings that MSH may not increase tyrosinase activity only through alterations in enzyme synthesis, we plan to investigate the possibility that MSH may act by (1)\decreasing tyrosinase degradation rates, (2)\inducing the synthesis of a tyrosinase activator protein, or (3)\promoting the inactivation of a tyrosinase inhibitor. Double-labeling studies will be used to determine if the selective synthesis of any non-tyrosinase proteins is stimulated by MSH. We will also determine if MSH alters mRNA levels for any specific proteins by analyzing proteins translated from mRNA derived from control and MSH-treated cultures and translated in an in vitro reticulocyte translation system. The effect of cAMP dependent protein kinase on the phosphorylation and activity of purified tyrosinase and melanosome-bound tyrosinase will be assessed. We will continue our investigation of the possible modulatory effects of steroid hormones, insulin, retinoids, calcium and calmodulin on the cAMP regulation of tyrosinase activity.